CN111808173B - Polypeptide-quantum dot compound, preparation method and application thereof - Google Patents
Polypeptide-quantum dot compound, preparation method and application thereof Download PDFInfo
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- CN111808173B CN111808173B CN201910293122.7A CN201910293122A CN111808173B CN 111808173 B CN111808173 B CN 111808173B CN 201910293122 A CN201910293122 A CN 201910293122A CN 111808173 B CN111808173 B CN 111808173B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/88—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing selenium, tellurium or unspecified chalcogen elements
- C09K11/881—Chalcogenides
- C09K11/883—Chalcogenides with zinc or cadmium
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention provides a polypeptide-quantum dot compound, wherein in the compound, a polypeptide and quantum dots are combined with each other, the amino acid sequence of the polypeptide is SEQ ID NO.1, the N end of the polypeptide is modified with Biotin Biotin, the surface of the quantum dots is modified with streptavidin, and the streptavidin is combined with the Biotin Biotin modified with the N end of the polypeptide. Also provides a preparation method and application thereof. The polypeptide provided by the invention can be specifically combined with a chemokine receptor CXCR4 which is highly expressed by tumor cells; meanwhile, compared with quantum dots, the polypeptide-quantum dot compound has strong affinity to tumor cells with high CXCR4 expression, provides a feasible method and technical means for targeted treatment of cancers, and has important value in targeted treatment of tumors.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a functionalized quantum dot compound, and especially relates to an application of a polypeptide-quantum dot compound capable of targeting cell surface receptors in detecting and inhibiting tumor metastasis.
Background
Cancer is one of the major causes of death in humans today, severely affecting human health. In recent years, there has been a deep search from the pathogenesis of cancer to diagnostic and therapeutic means in order to increase the survival rate of tumor patients. Many studies have found that the chemotaxis CXCL12/CXCR4 plays an important role in the tumorigenesis, progression and metastasis. It is reported in the literature that cells express CXCR4 in at least 23 different types of tumor tissue, and thus this chemokine receptor CXCR4 can be used as an entry point for tumor studies. According to the existing research results in many laboratories, chemokine receptor antagonists can inhibit macrophage infiltration, induce tumor growth arrest or apoptosis, prevent metastatic spread, and block the interaction between CXCL12/CXCR4 through a targeting agent to realize tumor treatment. Among the many classes of targeting agents, polypeptides are of interest because of their small size, availability via large scale synthetic procedures, and low immunogenicity. The subject group designs an E5 polypeptide, and experiments prove that the E5 polypeptide has high affinity with a chemokine receptor CXCR4 on the cellular level of leukemia, breast cancer and the like, can be specifically combined with the CXCR4 and has the function of inhibiting the migration capability of CXCR4 high-expression tumor cells, and is a polypeptide targeting agent which can be widely applied to tumor detection and treatment. Compared with the traditional material, the nano material has unique superiority in the aspects of drug transportation, diagnosis and biological imaging, and brings new thought for anti-tumor research. The nano material can improve the curative effect of the chemotherapeutic drugs, reduce the toxicity of the systemic biological drugs, realize the targeted delivery of the drugs, improve the bioavailability of the drugs and the like. The application of nanomaterials brings hopes for tumor treatment. As a spherical inorganic nano material, the Quantum Dots (QDs) have the advantages of easy control of excitation and emission wavelength, high Quantum yield, easy modification, no fluorescence bleaching and the like. Compared with the traditional fluorescent probe, the quantum dot has wide absorption spectrum, narrow emission spectrum and large Stokes shift in the aspect of biological imaging, and can image deep tissues in a near infrared region; but the use of quantum dots is limited in biocompatibility and cytotoxicity. The surface of the quantum dot is modified by the polypeptide targeting agent E5, and the formed novel E5 polypeptide-quantum dot compound system combines the advantages and functions of the polypeptide targeting agent and the quantum dot, and realizes the targeting function of the quantum dot while improving the biocompatibility of the quantum dot. Provides a feasible treatment method for tumor detection and tumor metastasis inhibition.
Disclosure of Invention
The invention aims to provide a polypeptide-quantum dot compound system with a targeting function and high detection sensitivity, and a preparation method and application thereof. The quantum dot has high luminous stability and luminous efficiency, and the combined E5 polypeptide has high affinity with tumor cells highly expressing CXCR4 receptor, so as to construct a polypeptide-quantum dot compound with targeting function.
To achieve the above object, in a first aspect of the present invention, there is provided a polypeptide-quantum dot complex, wherein a polypeptide and a quantum dot are combined with each other, the amino acid sequence of the polypeptide is SEQ ID NO.1, the N-terminal of the polypeptide is modified with Biotin Biotin, the surface of the quantum dot is modified with streptavidin, and the streptavidin is combined with the N-terminal modified Biotin Biotin of the polypeptide.
The quantum dots are selected from one or more of the following nano materials: cdSe, cdTe, cdS, znS, znSe, cdSe/ZnS, inP, inGaP; preferably CdSe/ZnS, most preferably spherical CdSe/ZnS.
The complex according to the first aspect of the present invention, wherein the molar ratio of the polypeptide to the quantum dot in the complex is 100-1:1, preferably 10-1:1, most preferably 10:1.
A second aspect of the present invention provides a method of preparing a complex according to the first aspect, the method comprising the steps of:
(1) Respectively preparing a quantum dot solution and a polypeptide solution;
(2) Uniformly mixing and incubating the quantum dot solution and the polypeptide solution obtained in the step (1), and obtaining a polypeptide-quantum dot compound solution;
(3) And (3) centrifuging the solution obtained in the step (2), and removing the supernatant to obtain the purified polypeptide-quantum dot compound.
According to the preparation method of the second aspect of the present invention, wherein in the step (1), the preparation method of the polypeptide solution comprises the steps of: the polypeptide is dissolved in ultrapure water to prepare a polypeptide solution with a concentration of 0.1-10 mM, preferably 1mM, and then diluted with boric acid buffer solution to prepare a polypeptide solution with a concentration of 1-100. Mu.M, preferably 10. Mu.M.
The preparation method according to the second aspect of the present invention, wherein in the step (2), the incubation temperature is 20 to 40 ℃; and/or
The incubation time is 0.5-12 h.
The production method according to the second aspect of the present invention, wherein, in the step (3), the centrifugal speed is not less than 20000rpm.
In a third aspect, the present invention provides a polypeptide targeting agent comprising: the polypeptide-quantum dot complex of the first aspect and/or the polypeptide-quantum dot complex produced according to the method of the second aspect.
In a fourth aspect the present invention provides the use of a polypeptide-quantum dot complex of the first aspect or a polypeptide-quantum dot complex prepared according to the method of the second aspect in the manufacture of a medicament for the treatment of a tumour and/or a tumour detection product.
In a fifth aspect, the invention provides the use of a polypeptide-quantum dot complex of the first aspect or a polypeptide-quantum dot complex prepared according to the method of the second aspect in the preparation of a product for inhibiting the ability of CXCR4 to highly express tumor cells to migrate.
The invention aims to provide a polypeptide-quantum dot composite system with a targeting function and high detection sensitivity and application thereof. The quantum dot has high luminous stability and luminous efficiency, and the combined E5 polypeptide has high affinity with tumor cells highly expressing CXCR4 receptor, so as to construct a polypeptide-quantum dot compound with targeting function.
Aiming at the purposes, the technical scheme provided by the invention is as follows:
the invention provides an application of a polypeptide-quantum dot compound with high affinity to a cell surface receptor CXCR4 in tumor detection, wherein the amino acid sequence of the polypeptide (E5) is SEQ ID NO.1, and the E5 polypeptide (N-terminal modified Biotin) has high affinity with a cell surface chemokine receptor (CXCR 4); the quantum dots are CdSe/ZnS nano materials, and Streptavidin (SA) modified on the surfaces of the quantum dots can react with Biotin at the N end of E5 polypeptide to form strong non-covalent chemical bonds, so that an E5 polypeptide-quantum dot compound system is obtained. The invention also provides a preparation method of the E5 polypeptide-quantum dot compound, which comprises the following steps:
1) Respectively preparing quantum dots and polypeptide solution;
2) Uniformly mixing the prepared quantum dots and the polypeptide solution to obtain a polypeptide-quantum dot compound solution;
3) And centrifuging the solution, and removing the supernatant to obtain the purified polypeptide-quantum dot compound.
In the above method, in step 1), the polypeptide is dissolved in ultrapure water to prepare a polypeptide solution having a concentration of 1mM, and then diluted with boric acid buffer to prepare a polypeptide solution having a concentration of 10. Mu.M.
Wherein, the molar ratio of the polypeptide to the quantum dots is 10-1:1, preferably 10:1.
In the method, the incubation temperature in the step 2) is 20-40 ℃ and the incubation time is 0.5-12 h.
The aforementioned method, wherein the centrifugation speed used in step 3) exceeds 20000rpm, is used to remove excess polypeptide in the polypeptide-quantum dot complex solution. The invention also provides application of the polypeptide-quantum dot compound in tumor detection.
The polypeptide provided by the invention can be specifically combined with a chemokine receptor CXCR4 which is highly expressed by tumor cells, and meanwhile, compared with quantum dots, the polypeptide-quantum dot compound has strong affinity to the tumor cells which are highly expressed by CXCR4, thereby providing a feasible method and technical means for targeted treatment of cancers and having important value in targeted treatment of tumors.
The polypeptide-quantum dot complexes of the invention may have, but are not limited to, the following beneficial effects:
the polypeptide provided by the invention can be specifically combined with a chemokine receptor CXCR4 which is highly expressed by tumor cells; meanwhile, compared with quantum dots, the polypeptide-quantum dot compound has strong affinity to tumor cells with high CXCR4 expression, provides a feasible method and technical means for targeted treatment of cancers, and has important value in targeted treatment of tumors.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a graph of a spherical GdSe/ZnS quantum dot electron microscope labeled with Streptavidin (SA) in example 1 of the present invention.
FIG. 2 is a graph showing the results of binding force measurement of the polypeptide of example 3 to Hela and Hek293t (a) cells at different polypeptide concentrations, respectively, and FIG. 2b is a graph showing the affinity of the polypeptide to Hela, hek293t and MDA-MB-231 at 10. Mu.M.
FIG. 3 is a graph showing the results of affinity of polypeptide-quantum dot complexes at different concentrations with Hela, hek293t and MDA-MB-231 according to example 4 of the present invention.
FIG. 4 is a confocal laser microscope image of the binding force of the quantum dot and polypeptide-quantum dot complex on the cell surface of Hela (a) and MDA-MB-231 (b) in example 4 of the present invention.
FIG. 5 is a graph showing the results of the proliferation-toxicity test of the polypeptide-quantum dot complex of example 5 of the present invention on the CXCR4 negative cell line breast cancer cell line MDA-MB-231, the human kidney epithelial cell line Hek293t and the CXCR4 positive cell line human cervical cancer cell line Hela.
Detailed Description
The invention is further illustrated by the following specific examples, which are, however, to be understood only for the purpose of more detailed description and are not to be construed as limiting the invention in any way.
This section generally describes the materials used in the test of the present invention and the test method. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. It will be apparent to those skilled in the art that in this context, the materials and methods of operation used in the present invention are well known in the art, if not specifically described.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
The breast cancer cell line MDA-MB-231, the human kidney epithelial cell line Hek293t and the human cervical cancer cell line Hela used in the following examples were all purchased from the national academy of medicine.
The aqueous solutions used in the examples below refer to sterile ultrapure water solutions, with the water quality parameter being a resistivity of 18.2 M.OMEGA.cm@25deg.C, unless otherwise specified.
Unless otherwise indicated, the reagents used in the examples below were all analytically pure reagents.
The PBS solution used in the examples below was 1 XPBS solution, and was filtered through a 0.22 μm filter, unless otherwise specified.
The sodium borate-HCl buffer used was a solution of 10mM (pH 8).
The reagent purchase sources and instrument models used in the following examples are as follows:
(1) Reagent purchase source:
PBS buffer, DMEM medium, opti-MEM medium, fetal bovine serum, diabody, FITC-Biotin all purchased from Thermo Fisher Scientific;
streptavidin-labeled quantum dots Qots-SA were purchased from beijing na crystal biotechnology limited;
4% paraformaldehyde solution was purchased from beijing soleba technologies limited;
CXCR4 antibodies were purchased from Biolegend;
MTS reagent detection kit was purchased from Promega.
(2) Instrument model:
a pure water meter (Merck Millipore, germany, model Milli-Q integrate 3);
nano particle size potentiometric analyzer (uk malva, model Zetasizer Nano ZS);
centrifuge (Beijing Lei Boer centrifuge Co., ltd., model LD 5-2A);
flow cytometry (BD biosciences model BD accuri C6);
continuous spectrum multifunctional enzyme labeling instrument (us Molecular Devices model SpectraMax i 3);
single photon laser confocal imager (Zeiss, model Zeiss 710, germany).
Example 1 preparation of E5 polypeptide-Quantum dots specifically binding to chemokine receptor CXCR4
The amino acid sequence of the polypeptide is Biotin-GGRSFFLLRRIQGCRFRNTVDD (synthesized by Anhui province national pharmaceutical industry Co., ltd., purity 98%), and a mother solution with a proper concentration is prepared before the experiment.
The quantum dot is GdSe/ZnS with spherical structure marked by Streptavidin (SA), the structure of the quantum dot is shown in figure 1 (synthesized by Beijing Najingsu Biotechnology Co., ltd.) and is prepared into mother solution with proper concentration before experiments.
Streptavidin (SA) has the function of combining with Biotin (Biotin), belongs to a common technical means, and has no substantial influence on the functions of the polypeptide.
Polypeptide dissolution: dissolving polypeptide powder with ultrapure water to obtain mother solution with concentration of 1mM, vortex oscillating for 3min to ensure polypeptide to be fully dissolved and dispersed, and storing at 4deg.C for use;
polypeptide-quantum dot preparation: polypeptide stock was diluted to 10 μm with sodium borate buffer following polypeptide: and adding the quantum dots with corresponding volumes in a ratio of the concentration ratio of the quantum dots to 10:1, uniformly mixing, and incubating at 37 ℃ for 0.5 hour to obtain the polypeptide-quantum dot compound solution.
Polypeptide-quantum dot purification: centrifuging the polypeptide-quantum dot compound solution at a speed exceeding 20000rpm for 1 hour, and removing the supernatant to obtain the purified polypeptide-quantum dot compound.
EXAMPLE 2 validation of CXCR4 positive and negative cell lines
1. Cell material used
Human cervical cancer cell line Hela is used as CXCR4 positive cell to be verified, breast cancer cell line MDA-MB-231 and human renal epithelial cell line Hek293t is used as CXCR4 negative cell to be verified.
2. Specific method
1) Polypeptide dissolution: dissolving polypeptide powder with ultrapure water to obtain mother solution with concentration of 1mM, vortex oscillating for 3min to ensure polypeptide to be fully dissolved and dispersed, and storing at 4deg.C for use.
2) Cell count: culturing Hela, MDA-MB-231 and Hek293t cells according to a cell culture method, digesting and centrifuging the cells when the cells grow to 80-90% of the total culture dish and are connected into a net shape, discarding the culture solution, adding PBS for resuspension, and counting after being gently blown and beaten uniformly.
3) Sample preparation:
incubating the polypeptide: collecting a certain amount (more than or equal to 106) of cells in a 1.5mL centrifuge tube, diluting polypeptide mother liquor to 10 mu M, adding 50 mu L of the polypeptide into the centrifuge tube with the cells collected, gently beating the cells to be uniformly suspended by a pipetting gun, incubating for 2h at 37 ℃, centrifuging the incubated cell suspension in a centrifuge with the rotation speed of 2000rpm for 5min, discarding the supernatant, re-suspending by PBS, repeating the steps for three times, removing non-specifically bound polypeptide, adding Biotin-FITC re-suspending cells diluted by PBS, incubating for 1h at 4 ℃, centrifuging to remove the supernatant, re-suspending by PBS to 200 mu L of solution, and filtering to obtain a flow type test solution of the cells;
incubating the antibody: the antibody and the polypeptide are incubated with cells in the same volume, the antibody mother solution diluent (the control group is the isotype control) is taken to be mixed with the cells to be blown uniformly, the incubated cell suspension is centrifuged for 5min in a centrifuge with the rotating speed of 2000rpm at the temperature of 4 ℃ for 1h, the supernatant is discarded, the PBS is used for resuspension, the non-specific binding antibody is removed in three times, then the cells are resuspended into 200 mu L of solution, and the flow type test solution of the cells is obtained after filtration.
4) Flow cytometer measurement: the FL-1 channel (FITC fluorescence detection channel, excitation wavelength 488 nm) of a flow cytometer (FCM, BD Accuri) is selected for detection; firstly, detecting flow type test solution of cells in a control group, adjusting parameters to enable the positive binding rate of the control group to be close to 0, sequentially detecting test sample test solution of an experiment group of corresponding cell lines by taking the positive binding rate as a reference standard, and recording the binding rate of polypeptides and antibodies to the cells.
The results of the three cell lines are shown in Table 1.
TABLE 1 detection results of cell lines
By combining the detection results of the antibodies, the Hela cells are CXCR4 positive cells, the MDA-MB-231 and the Hek293t are CXCR4 negative cells, which are consistent with the prior reports, and the polypeptide can specifically recognize CXCR4.
Example 3 E5 polypeptide targeting validation
1. Cell lines used
The confirmed and reported Hela cells were CXCR4 positive cells, and MDA-MB-231 and Hek293t were CXCR4 negative cells.
2. Specific method
1) Polypeptide dissolution: dissolving polypeptide powder with ultrapure water to obtain mother solution with concentration of 1mM, vortex oscillating for 3min to ensure polypeptide to be fully dissolved and dispersed, and storing at 4deg.C for use.
2) Cell count: culturing Hela and Hek293t cells according to a cell culture method, digesting and centrifuging the cells when the cells grow to 80-90% of full culture dishes and are connected into a net shape, discarding culture solution, adding PBS for resuspension, lightly blowing and uniformly counting.
3) Sample preparation: a certain amount (more than or equal to 106) of cells are collected in a 1.5mL centrifuge tube, and polypeptide mother liquor is diluted to the following concentration gradient: 0.1, 2, 4, 8 and 10 mu M, wherein the control group is not added with polypeptide but the subsequent steps are consistent, 50 mu L of the control group is added into a centrifugal tube for collecting cells, the cells are gently beaten by a pipetting gun until the cells are uniformly suspended, the culture is carried out for 2 hours at 37 ℃, the incubated cell suspension is centrifuged for 5 minutes in a centrifugal machine with the rotating speed of 2000rpm, the supernatant is discarded, the PBS is used for resuspension, the non-specific binding polypeptide is removed by repeating the steps for three times, then the Biotin-FITC diluted by PBS is added for resuspension of the cells, the culture is carried out for 1 hour at 4 ℃, the supernatant is centrifuged, the PBS is used for resuspension into 200 mu L of solution, and the flow type test liquid of the cells is obtained after filtration.
4) Flow cytometer measurement: the FL-1 channel (FITC fluorescence detection channel, excitation wavelength 488 nm) of a flow cytometer (FCM, BD Accuri) is selected for detection; firstly, detecting the flow type test solution of the cells of the control group, adjusting parameters to enable the positive binding rate of the control group to be close to 0, sequentially detecting the test solution of the test group sample of the corresponding cell line by taking the positive binding rate as a reference standard, and recording the fluorescence intensity of the binding of the polypeptide and the cells, wherein the result is shown in figure 1.
From fig. 2a, it can be seen that the polypeptide has a high affinity for CXCR4 positive cell Hela, whereas it has a low affinity for CXCR4 negative cell Hek293 t. The same experimental procedure was further followed to detect MDA-MB-231, a CXCR4 negative cell, and the polypeptide was found to have very low affinity for this cell, as shown in FIG. 2 b.
Example 4 E5 polypeptide-Quantum dot Complex targeting validation
1. Cell lines used
The confirmed and reported Hela cells were CXCR4 positive cells, and MDA-MB-231 and Hek293t were CXCR4 negative cells.
2. Flow cytometer detection
1) Preparing mother liquor:
polypeptide dissolution: dissolving polypeptide powder with ultrapure water to obtain mother solution with concentration of 1mM, vortex oscillating for 3min to ensure polypeptide to be fully dissolved and dispersed, and storing at 4deg.C for use;
polypeptide-quantum dot preparation: polypeptide stock was diluted to 10 μm with sodium borate buffer following polypeptide: and adding quantum dots with corresponding volumes into the solution with the concentration ratio of the quantum dots being 10:1, uniformly mixing, and incubating at room temperature for 30min to obtain the polypeptide-quantum dot compound solution.
2) Cell count: culturing Hela, MDA-MB-231 and Hek293t cells according to a cell culture method, digesting and centrifuging the cells when the cells grow to 80-90% of the total culture dish and are connected into a net shape, discarding the culture solution, adding PBS for resuspension, and counting after being gently blown and beaten uniformly.
3) Sample preparation: a certain amount (more than or equal to 106) of cells are collected in a centrifuge tube with the concentration of 1.5mL, and the polypeptide-quantum dot complex solution is diluted to the following concentration: 50nM and 100nM, the control group is the same concentration of quantum dot solution but the subsequent steps are consistent, 50 μL is taken and added to a centrifuge tube where cells are collected, the cells are gently beaten to be uniformly suspended by a pipette, incubated for 2h at 4 ℃, the incubated cell suspension is centrifuged for 5min in a centrifuge with the rotation speed of 2000rpm, the supernatant is discarded, and then resuspended by PBS, the non-specifically bound polypeptide-quantum dot complex is removed, 200 μL of solution is resuspended by PBS, and the flow test solution of the cells is obtained after filtration.
4) Flow cytometer measurement: the FL-2 channel (PE fluorescence detection channel, excitation wavelength 488 nm) of a flow cytometer (FCM, BD Accuri) is selected for detection; firstly, detecting the flow type test solution of the cells of the control group, adjusting parameters to enable the positive binding rate of the control group to be close to 0, sequentially detecting the test solution of the test group sample of the corresponding cell line by taking the positive binding rate as a reference standard, and recording the fluorescence intensity of the binding of the polypeptide and the cells, wherein the result is shown in figure 3.
As can be seen from fig. 3, after the polypeptide is modified to the surface of the quantum dot by streptavidin and biotin, the targeting characteristic of the polypeptide is not changed due to the basic sequence of the polypeptide, the binding capacity of the polypeptide is increased along with the increase of the concentration of the polypeptide-quantum dot, and the binding capacity of the polypeptide is very low for CXCR4 negative cells MDA-MB-231 and Hek293 t. The polypeptide-quantum dot complex is consistent with the binding of the cell line, which indicates that the targeting of the polypeptide is not changed by the introduced quantum dot, and the polypeptide-quantum dot complex can detect CXCR4 positive cells at a lower concentration due to the high quantum yield of the quantum dot.
2. Single photon laser confocal imaging detection
1) Cell inoculation: preparing cell suspension, counting, adding 1×104 cells into Confocal dish, and uniformly attaching to bottom to obtain final product.
2) Preparing mother liquor:
polypeptide dissolution: dissolving polypeptide powder with ultrapure water to obtain mother solution with concentration of 1mM, vortex oscillating for 3min to ensure polypeptide to be fully dissolved and dispersed, and storing at 4deg.C for use;
polypeptide-quantum dot preparation: polypeptide stock was diluted to 10 μm with sodium borate buffer following polypeptide: and adding the quantum dots with corresponding volumes in a ratio of the quantum dot concentration ratio of 10:1, uniformly mixing, and incubating at room temperature for 37 minutes to obtain the polypeptide-quantum dot compound solution.
3) Sample preparation: the polypeptide-quantum dot solution and quantum dot stock solution were diluted to 100nM with opti-MEM medium, respectively.
4) Cell incubation: the prepared sample solution was incubated with Hela cells and MDA-MB-231 cells of the Confocal dish at 4℃for 2h, respectively, and then washed twice with PBS buffer.
5) Cell fixation: after the cells in step 4) were washed clean, 200. Mu.L of 4% paraformaldehyde was added to each dish, and the mixture was fixed for 15min, followed by washing twice with PBS buffer.
6) Cell nuclear staining: after the cells in step 5) were washed clean, 200. Mu.L of Hochest 33324 nuclear reagent at a concentration of 1. Mu.g/mL was added to each dish, stained for 15min, then washed twice with PBS buffer, and single-molecule laser confocal observation was performed by adding 500. Mu.L of medium or PBS buffer.
7) Confocal laser microscopy imaging: the software is respectively provided with a bright field channel, a host 33324 channel and a Quantum dots 605 channel, after a cell plane is found, the cell definition is adjusted, the properties of each channel are sequentially adjusted, and the photo is taken, and the result is shown in figure 4.
As can be seen from fig. 4, the targeting function of the polypeptide is not changed after the polypeptide is attached to the quantum dot, and the polypeptide-quantum dot complex still has high affinity to CXCR4 high-expression cells, which is consistent with the previous flow results.
Example 5 E5 polypeptide-Quantum dot pair breast cancer cell lines MDA-MB-231, human renal epithelial cell lines Hek293t and
assay for human cervical cancer cell line Hela cytotoxicity
Cells in the logarithmic growth phase were harvested for cytotoxicity assays. Cells were counted by digestion, 5X 103 cells were cultured in 96-well plates (Corning) each using DMEM medium (containing one tenth of fetal calf serum and one hundredth of a double antibody), the 96-well plates were pre-incubated in incubator at 37℃under 5% CO2 for 24h, and the 96-well plates were exchanged with polypeptide-quantum dot solutions of different concentrations prepared with the medium every day. The 96-well plate was placed in an incubator for culturing for 24 hours, then the medium was discarded, 100. Mu.L opti-MEM and 20. Mu.L MTS solution were added to each well, incubation was continued in the incubator for 2 hours, and absorbance (OD) values at 490nm were measured with a continuous spectrum multifunctional microplate reader (SpectraMax i3, molecular Devices, USA), and cell viability was calculated.
Cell viability = OD490nm (polypeptide-quantum dot)/OD 490nm (blank control)
Cell viability under polypeptide-quantum dot incubation conditions is shown in figure 5. As can be seen from the cell viability of fig. 5, the targeting polypeptide-quantum dots were not cytotoxic to both CXCR4 positive and CXCR4 negative cell lines, and were well biocompatible.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes may be made in the individual conditions without departing from the spirit and scope of the invention. It is to be understood that the invention is not to be limited to the described embodiments, but is to be given the full breadth of the claims, including equivalents of each of the elements described.
Sequence listing
<110> national center of nanoscience
<120> polypeptide-quantum dot complex, preparation method and application thereof
<130> YZDI-190018
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
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<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Gly Gly Arg Ser Phe Phe Leu Leu Arg Arg Ile Gln Gly Cys Arg Phe
1 5 10 15
Arg Asn Thr Val Asp Asp
20
Claims (7)
1. The polypeptide-quantum dot compound is characterized in that in the compound, a polypeptide and quantum dots are combined with each other, the amino acid sequence of the polypeptide is SEQ ID NO.1, the N end of the polypeptide is modified with Biotin Biotin, the surface of the quantum dots is modified with streptavidin, and the streptavidin is combined with the Biotin Biotin modified with the N end of the polypeptide;
the molar ratio of the polypeptide to the quantum dots is 10:1;
the quantum dots are spherical CdSe/ZnS.
2. A method of preparing a composite according to claim 1, characterized in that the method comprises the steps of:
(1) Respectively preparing a quantum dot solution and a polypeptide solution;
(2) Uniformly mixing and incubating the quantum dot solution and the polypeptide solution obtained in the step (1), and obtaining a polypeptide-quantum dot compound solution;
(3) And (3) centrifuging the solution obtained in the step (2), and removing the supernatant to obtain the purified polypeptide-quantum dot compound.
3. The method according to claim 2, wherein in the step (1), the method for preparing the polypeptide solution comprises the steps of: the polypeptide is dissolved by ultrapure water to prepare a polypeptide solution with the concentration of 0.1-10 mM, and then the polypeptide solution is diluted by boric acid buffer solution to prepare a polypeptide solution with the concentration of 1-100 mu M.
4. A method according to claim 3, wherein in step (1), the method of preparing the polypeptide solution comprises the steps of: the polypeptide was dissolved in ultrapure water to prepare a polypeptide solution having a concentration of 1mM, and then diluted with boric acid buffer to prepare a polypeptide solution having a concentration of 10. Mu.M.
5. The method according to claim 2, wherein in step (2), the incubation temperature is 20 to 40 ℃; and/or
The incubation time is 0.5-12 h.
6. The method according to claim 2, wherein in the step (3), the centrifugal speed is not less than 20000rpm.
7. A polypeptide targeting agent, said polypeptide targeting agent comprising: the polypeptide-quantum dot complex of claim 1 and/or the polypeptide-quantum dot complex produced according to the method of any one of claims 2 to 6.
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| WO2016011878A1 (en) * | 2014-07-23 | 2016-01-28 | 国家纳米科学中心 | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method therefor and application thereof |
| CN104231050A (en) * | 2014-09-25 | 2014-12-24 | 国家纳米科学中心 | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method for polypeptide complex and application of polypeptide and polypeptide complex |
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